Isolation of a highly quiescent subpopulation of primitive leukemic cells in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is characterized by an increased proliferati ve activity of the leukemic progenitors that produce an elevated number of mature granulocytes. Nevertheless, cell cycle-active agents, even in very h igh doses, are alone unable to eradicate the leukemic clone, suggesting the presence of a rare subset of quiescent leukemic stem cells. To isolate suc h cells, we first used Hoechst 33342 and Pyronin Y staining to obtain viabl e G(0) and G(1)/S/G(2)/M fractions of CD34(+) cells by fluorescence-activat ed cell sorting (FACS) from 6 chronic-phase CML patients' samples and confi rmed the quiescent and cycling status of the 2 fractions by demonstration o f expected patterns of Ki-67 and D cyclin expression. Leukemic (Ph+/BCR-ABL (+)) cells with in vitro progenitor activity and capable of engrafting immu nodeficient mice were identified in the directly isolated Go cells. Single- cell reverse transcriptase-polymerase chain reaction (RT-PCR) analysis show ed that many leukemic CD34(+) G(0) cells also expressed BCR-ABL mRNA, CD34( +) from 8 CML patients were also labeled with carboxyfluorescein diacetate succinimidyl diester (CFSE) before being cultured (with and without added g rowth factors) to allow viable cells that had remained quiescent tie, CFSE) after 4 days to be retrieved by FAGS, Leukemic progenitors were again det ected in all quiescent populations isolated by this second strategy, includ ing those exposed to a combination of flt3-ligand, Steel factor, interleuki n-3, interleukin-6, and granulocyte colony-stimulating factor, These findin gs provide the first direct and definitive evidence of a deeply but reversi bly quiescent subpopulation of leukemic cells in patients with CML with bot h in vitro and in vivo stem cell properties. (C) 1999 by The American Socie ty of Hematology.